Methods of treatment and related compositions

ABSTRACT

The present invention relates to methods of treating a disease characterised by aberrant cell proliferation (e.g., a cancer) in a human subject in need thereof. In particular, the present invention relates to treating the above conditions by administering a therapeutically effective amount of at least one agent that increases activation of a receptor of at least one type II interferon and/or type I interferon, and administering to the subject at least one agent that inhibits the Hedgehog (Hh) signalling pathway (e.g., Vismodegib). Also provided are pharmaceutical compositions, including controlled release pharmaceutical compositions, containing at least one agent that increases activation of a receptor of at least one type II interferon and/or type I interferon (e.g., a checkpoint inhibitor), an inhibitor of Hh signalling pathway, and a controlled release matrix such as a SiO 2  matrix gel.

The present application is a U.S. National Stage Application under 35U.S.C. § 371 of International Application No. PCT/AU2021/050167 filedFeb. 26, 2021, which claims the benefit of priority to Australian PatentApplication No. 2020900586 filed Feb. 28, 2020 and Australian PatentApplication No. 2020900813 filed Mar. 17, 2020, the disclosures of whichare hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The specification relates generally to the field of therapy for diseasescharacterised by aberrant cell proliferation.

BACKGROUND OF THE INVENTION

The effective treatment of conditions characterised by aberrant cellproliferation, e.g., cancers, remains an enormous challenge toclinicians. Inhibitors of the Hedgehog (Hh) signalling pathway, e.g.,Vismodegib, have become a valuable addition to the arsenal of agentsavailable for treating cancer and other proliferative disorders.Nevertheless, the development of resistance, or, more commonly,persistence in response to Hh signalling inhibitors diminishes theirusefulness as a monotherapies. Further, the extended treatment generallyrequired for treatment with Hh signalling inhibitors and theirassociated adverse effects on patients present further disadvantages forthe use of Hh signalling inhibitors for treating proliferativedisorders.

Thus, there is an ongoing need for therapeutic methods and compositionsthat can potentiate the therapeutic efficacy of inhibitors of the Hhsignalling pathway.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that a combination of oneor more inhibitors of the Hh signalling pathway (e.g., Vismodegib) incombination with an agent that increases activation of a receptor of atleast one type II interferon and/or type I interferon synergisticallyinduces a more complete elimination of aberrantly proliferating cellsand reduces the required dosing level and treatment duration requiredfor therapeutically effective use of inhibitors of the Hh signallingpathway.

Accordingly, provided herein is a method of treating a subject sufferingfrom a disease characterised by aberrant cell proliferation, the methodcomprising administering to the subject a therapeutically effectiveamount of: (i) at least one agent that increases activation of areceptor of at least one type II interferon and/or type I interferon;and (ii) at least one agent that inhibits the Hedgehog (Hh) signallingpathway.

In some embodiments the subject to be treated is a mammal. In somepreferred embodiments the subject to be treated is a human.

In some embodiments the at least one agent in (i) is a type IIinterferon and/or type I interferon, or a polynucleotide encoding a typeII interferon and/or type I interferon, or an agonist for a receptor ofat least one type II interferon and/or type I interferon.

In some embodiments the at least one agent in (i) comprises at least onetype II interferon and/or type I interferon, or a polynucleotideencoding a type II interferon and/or type I interferon, or an agonistfor a receptor of at least one type II interferon and/or type Iinterferon. In some embodiments the method includes administering atherapeutically effective amount of a type II interferon to the subject.In some embodiments, where a type II interferon is to be administered,the type II interferon is interferon gamma. In other embodiments the atleast one agent in (i) is a polynucleotide for expression of a type IIinterferon and/or type I interferon in the subject to be treated. Insome preferred embodiments, the polynucleotide is a polynucleotide forexpression of interferon gamma.

In some preferred embodiments the polynucleotide for expression of thetype II interferon and/or type I interferon is encoded by a recombinantvirus administered to the subject. In some embodiments the recombinantvirus is a recombinant DNA virus. In some embodiments the recombinantDNA virus is an adenovirus, an adeno-associated virus (AAV), a herpessimplex virus (HSV), or a lentivirus. In some preferred embodiments therecombinant DNA virus is an adenovirus. In some preferred embodimentsthe recombinant adenovirus is for expression of interferon gamma. Insome preferred embodiments the adenovirus for expression of interferongamma is ASN-002/SP-002. In some embodiments, where the method includesadministering a recombinant virus for expression of the therapeuticallyeffective amount of the type II interferon and/or a type I interferon,the recombinant virus is administered as at least 1×10⁸ viral particles(vp) per lesion per dosing day.

In some embodiments the at least one agent in (i) is an agent thatstimulates endogenous production and/or release of a type II interferonand/or type I interferon in the subject. In some embodiments the atleast one agent in (i) is an agent that stimulates endogenous productionand/or release of interferon gamma in the subject. In some embodimentsthe agent that stimulates endogenous production and/or release of a typeII interferon and/or type I interferon includes one or more checkpointinhibitors. In some embodiments the one or more checkpoint inhibitorsinhibit activation of the PD-1 receptor, the CTLA-4 receptor, or boththe PD-1 receptor and the CTLA-4 receptor.

In some preferred embodiments the one or more checkpoint inhibitorsinclude a polypeptide checkpoint inhibitor or a peptide checkpointinhibitor. In some embodiments the polypeptide checkpoint inhibitor orthe peptide checkpoint inhibitor binds to a checkpoint receptor. Inother embodiments the polypeptide checkpoint inhibitor or the peptidecheckpoint inhibitor binds to a checkpoint receptor ligand. In someembodiments the polypeptide checkpoint inhibitor or the peptidecheckpoint inhibitor binds to a checkpoint receptor ligand selected fromthe group consisting of: PD-L1, PD-L2, CD80/B7-1, CD86/B7-2, and anycombination thereof. In some embodiments the polypeptide checkpointinhibitor is an antibody or antigen-binding portion thereof. In someembodiments the antibody is is selected from the group consisting of:Ipilimumab, Pembrolizumab, Nivolumab, Atezolizumab, Avelumab,Durvalumab, Cemiplimab, AGEN1181, Tremelimumab, and any combinationthereof. In some embodiments the antibody or antigen-binding portionthereof is bispecific. In some embodiments, where the antibody orantigen-binding portion thereof is bispecific, the bispecific antibodyis selected from the group consisting of: M7824, MGD013, FS118,MCLA-134, XmAb-20717, ATOR-1015, and any combination thereof.

In some embodiments the one or more checkpoint inhibitors comprise atargeting polynucleotide. In some embodiments the the targetingpolynucleotide is selected from the group consisting of: siRNA, RNAi,antisense oligonucleotides, CRISPR guide RNAs (gRNAs), and anycombination thereof.

In some embodiments the at least one agent in (i) is administeredsystemically, intralesionally, or topically.

In some embodiments the at least one agent that inhibits the Hedgehog(Hh) signalling pathway inhibits the agent that inhibits the Hhsignalling pathway inhibits a target selected from among SMO, PTCH1,GLI, SHHat, tGLI1, and SHH. In some embodiments the Hh signallingpathway target is SMO. In other embodiments the Hh signalling pathwaytarget is SHH.

In some embodiments the agent that inhibits the Hh signalling pathway isa small molecule inhibitor. In some embodiments the small moleculeinhibitor is selected from the group consisting of: Vismodegib,Sonidegib, Saridegib, IPI 926, LEQ-506, Taladegib, Itraconazole,Glasdegib, Jervine, CUR61414, BMS-833923, TAK-441, MRT-92, GDC-0449,HH-13, GANT61, and HH-20. In some preferred embodiments the smallmolecule inhibitor is Vismodegib. In some embodiments the small moleculeinhibitor of the Hh signalling pathway is administered at a dose ofabout 150 mg to about 500 mg per day. In some embodiments the smallmolecule inhibitor of the Hh signalling pathway is administered at adose of about 150 mg/day.

In other embodiments the agent that inhibits the Hh signalling pathwaycomprises a polypeptide, a polynucleotide, or a peptide.

In some embodiments the agent that inhibits the Hh signalling pathwaycomprises an antibody or an antigen-binding portion thereof that bindsspecifically to an Hh signalling pathway target. In some embodiments theagent comprises an antigen-binding portion against SHH, SMO, PTCH1, GLI,SHHat, or tGLI1. In some embodiments the agent comprises an antibody oran antigen-binding portion thereof against SHH. In some embodiments theantibody or antigen-binding portion thereof against SHH is an antibodyor antigen-binding portion thereof selected from the group consistingof: 5E1, MEDI-5304, 1C11-2G4, antigen-binding portions thereof, andantibodies or antigen-binding portions thereof that compete with any oneof 5E1, MEDI-5304, or 1C11-2G4 for binding to SHH. In other embodimentsthe agent comprises an antibody or an antigen-binding portion thereofagainst PTCH1. In some embodiments the antibody against PTCH1 is theα-PTCH1 antibody.

In some embodiments the agent that inhibits the Hh signalling pathway isa polynucleotide. In some embodiments the polynucleotide encodes apolypeptide that inhibits the Hh signalling pathway. In someembodiments, the polynucleotide is or encodes a miRNA, an siRNA, anRNAi, or a CRISPR gRNA, an antisense RNA, or an antisenseoligonucleotide targeted against a Hh signalling pathway target.

In some embodiments, where the agent that inhibits the Hh signallingpathway is a polynucleotide, the subject is administered a recombinantvirus for expression of the polynucleotide. In some preferredembodiments the recombinant virus is a recombinant DNA virus.

In some embodiments the agent that inhibits the Hh signalling pathway isadministered systemically, intralesionally, or topically. In someembodiments the agent that inhibits the Hh signalling pathway isadministered systemically. In other embodiments the agent that inhibitsthe Hh signalling pathway is administered intralesionally. In someembodiments, irrespective of route of administration, the agent thatinhibits the Hh signalling pathway is administered as a controlledrelease formulation. In some embodiments, a controlled releaseformulation is administered through both intralesional and systemicroutes.

In some embodiments administration of (i) at least one agent thatincreases activation of a receptor of at least one type II interferonand/or type I interferon; and (ii) the agent that inhibits the Hedgehog(Hh) signalling pathway are performed separately during a first dosingperiod.

In some embodiments, during the first dosing period, (ii) isadministered after beginning administration of (i). In some embodiments,where (ii) is administered after beginning administration of (i), (ii)is administered at least one to three weeks after beginningadministration of (i). In other embodiments during the first dosingperiod, (ii) is administered before administration of (i). In someembodiments (ii) is administered at least one to three weeks beforebeginning administration of (i).

In other embodiments (i) at least one agent that increases activation ofa receptor of at least one type II interferon and/or type I interferon;and (ii) the agent that inhibits the Hedgehog (Hh) signalling pathwayare co-administered during a first dosing period.

In some embodiments, (i) and (ii) are co-administered in a singleformulation containing both (i) and (ii).

In some embodiments (i), (ii), or both (i) and (ii) are administeredmultiple times during a first dosing period.

In some embodiments (i) and (ii) are administered during a first dosingperiod and at least a second dosing period.

In some embodiments the subject is treated over a period of about oneweek to about twelve weeks. In some embodiments, where the subject istreated over a period of about one week to about twelve weeks, the doseof (ii) administered or treatment period with (ii) is limited to avoidinduction of at least one adverse event associated with treatment with(ii).

In some embodiments the disease characterised by aberrant cellproliferation is selected from the group consisting of: a cancer, afibrotic disease, or cutaneous warts. In some embodiments the diseasecharacterised by aberrant cell proliferation is a cancer. In someembodiments the cancer is selected from the group consisting of: basalcell carcinoma, melanoma, lymphoma, squamous cell carcinoma, Merkel cellcarcinoma, lung cancer, prostate cancer, sarcomas, medulloblastomas,cancers with desmoplastic stromas, colorectal cancer, ovarian cancer,breast cancer, gastric cancer, pancreatic cancer, mesothelioma,mesenchymal cancer, epithelial cancer, and adenocarcinomas. In somepreferred embodiments the cancer to be treated is a basal cell carcinoma(BCC). In some embodiments the subject suffering from BCC suffers fromBasal Cell Nevus Syndrome (BCNS) or sporadic BCC. In some embodiments,where the subject is to be treated for a cancer, the cancer is arecurrent cancer or a relapsing cancer.

In some embodiments at least two agents for inhibiting the Hh signallingpathway are administered. In some embodiments the at least two agentsinhibit different Hh signalling pathway targets.

Furthermore, the present invention provides a method of treating asubject suffering from a disease characterised by aberrant cellproliferation, the method comprising administering to the subject atherapeutically effective amount of: (i) at least one checkpointinhibitor; and (ii) at least one agent that inhibits the Hedgehog (Hh)signalling pathway.

In a further aspect provided herein is the use of at least one agentthat increases activation of a receptor of at least one type IIinterferon and/or type I interferon for the manufacture of a medicamentfor the treatment of disease characterised by aberrant cellproliferation in a subject, wherein the subject has been or will beadministered with at least one agent which inhibits Hedgehog (Hh)signalling pathway. In some embodiments the at least one agent thatincreases activation of the receptor comprises at least one type IIinterferon and/or type I interferon, a polynucleotide encoding a type IIinterferon and/or type I interferon.

In another aspect provided herein is the use of at least one least oneagent which inhibits the Hedgehog (Hh) signalling pathway for themanufacture of a medicament for the treatment of a disease characterisedby aberrant cell proliferation, in a subject, wherein the subject hasbeen or will be administered with at least one agent that increasesactivation of a receptor of at least one type II interferon and/or typeI interferon. In some embodiments the subject has been or will beadministered at least one type II interferon and/or type I interferon, apolynucleotide encoding the type II interferon and/or type I interferon,or an agonist for a receptor of at least one type II interferon and/ortype I interferon.

In another aspect provided herein is the use of at least one checkpointinhibitor for the manufacture of a medicament for the treatment ofdisease characterised by aberrant cell proliferation in a subject,wherein the subject has been or will be administered with at least oneagent which inhibits Hedgehog (Hh) signalling pathway.

In another aspect provided herein is the use of at least one least oneagent which inhibits the Hedgehog (Hh) signalling pathway for themanufacture of a medicament for the treatment of a disease characterisedby aberrant cell proliferation, in a subject, wherein the subject hasbeen or will be administered with at least checkpoint inhibitor.

In a further aspect provided herein is a pharmaceutical composition foruse in treatment of a disease characterised by aberrant cellproliferation, the pharmaceutical composition comprising: (i) at leastone agent that increases activation of a receptor of at least one typeII interferon and/or type I interferon; and (ii) at least one agentwhich inhibits the Hedgehog (Hh) signalling pathway. In some embodimentsthe pharmaceutical composition includes at least one type II interferonand/or type I interferon, or a polynucleotide encoding a type IIinterferon and/or type I interferon, or an agonist for a receptor of atleast one type II interferon and/or type I interferon. In someembodiments the pharmaceutical composition comprises a recombinant viruscomprising the polynucleotide encoding the type II interferon and/ortype I interferon for expression in a subject. In some preferredembodiments of the pharmaceutical composition the interferon isinterferon gamma.

In a related aspect provided herein is a pharmaceutical composition foruse in treatment of a disease characterised by aberrant cellproliferation, where the pharmaceutical composition includes: (i) atleast one checkpoint inhibitor; and (ii) at least one agent whichinhibits the Hedgehog (Hh) signalling pathway.

In some embodiments of any of the above-mentioned pharmaceuticalcompositions, the pharmaceutical composition also includes a controlledrelease matrix, wherein each of (i) and (ii) are interspersed throughoutthe controlled release matrix. In some embodiments the controlledrelease matrix comprises a SiO₂ matrix gel. In some embodiments the SiO₂matrix hydrogel comprises water and tetraethyl orthosilicate (TEOS) in afinal molar ratio of between about 5:1 to about 4,000:1. In someembodiments the ratio of water to TEOS is about 400:1.

In some embodiments the pharmaceutical compositions are depotformulations.

The steps, features, integers, compositions and/or therapeutic agentsdisclosed herein or indicated in the specification of this applicationindividually or collectively, and any combinations of two or more ofsaid steps or features.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (e.g. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1—Therapeutic response in B16F10 tumour-bearing mice to treatmentmonotherapy and combination treatment regimens with Vismodegib andAd5-mIFNgamma. A line graph of tumour volume over a treatment period intreatment regimens G1-G8 as indicated. Mice administered both Vismodegibat 50 mg/kg daily for seven days and intralesional Ad5-mIFNgamma (1×10¹⁰VPs/tumour) for three days (G4) exhibited the slowest tumour growth andhighest viability at day 20.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in viral vectorconstruction, transfection, gene knockdown, gene knockout, gene therapy,molecular genetics, cancer biology, cancer therapy, immunology,pharmacology, protein chemistry, and biochemistry).

Unless otherwise indicated, any recombinant molecular biology orimmunological techniques described herein are standard procedures, wellknown to those skilled in the art. Such techniques are described andexplained throughout the literature in sources such as, J. Perbal, APractical Guide to Molecular Cloning, John Wiley and Sons (1984), J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

As used herein, the term about, unless stated to the contrary, refers to+/−10%, more preferably +/−5%, of the designated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used in this application, the term “or” is intended to mean aninclusive “or” rather than an exclusive “or.” That is, unless specifiedotherwise, or clear from context, “X employs A or B” is intended to meanany of the natural inclusive permutations. That is, if X employs A; Xemploys B; or X employs both A and B, then “X employs A or B” issatisfied under any of the foregoing instances. Further, at least one ofA and B and/or the like generally means A or B or both A and B. Inaddition, the articles “a” and “an” as used in this application and theappended claims may generally be construed to mean “one or more” unlessspecified otherwise or clear from context to be directed to a singularform.

The term “disease characterised by aberrant cell proliferation” as usedherein, refers to any disease in which cell division is characterised bya diminished response, a lack of response, or an exaggerated response toone or more regulatory signals or proteins that modulate the rate ofcell division, cell survival, and/or cell death in a particularpopulation of cells. Examples of such regulatory signals include theregulatory signals or proteins include, but are not limited to, thepresence or absence of a growth factor, checkpoint proteins, tumoursuppressor proteins, pro-apoptotic proteins, and anti-apoptoticproteins. Examples of diseases characterised by aberrant cellproliferation include, but are not limited to, a cancer, a fibroticdisease, or cutaneous warts.

The term “type II interferon” as used herein, refers to interferons thatbind to the interferon gamma receptor, e.g., interferon gamma. In anembodiment, the type II interferon is a human interferon. “type IIinterferon” also includes pegylated forms of type II interferon.

The term “type I interferon” as used herein, refers to a subclass ofinterferons that bind to and activate the interferon alpha receptorcomplex. Non-limiting examples of type I interferons include: interferonalpha, interferon beta, interferon epsilon, interferon kappa, andinterferon omega 1. In an embodiment, the type I interferon is a humaninterferon. “type I interferon” also includes pegylated forms of type Iinterferon.

The term “agent that inhibits the Hedgehog (Hh) signalling pathway” asused herein, refers to any type of molecule that interferes with signaltransduction upstream or downstream of the canonical receptor Patched(PTCH1) and/or the co-receptors growth arrest-specific gene 1 (GAS1),cell adhesion associated oncogene regulated (CDO/CDON), and Brother ofCDO (BOC). Exemplary targets of inhibitors of Hh signalling pathwayinclude, but are not limited to, Patch 1 (PTCH1), smoothened (SMO),glioma-associated oncogene homolog (GLI), SHH acetyl transferase(SHHat), truncated GLI1 (tGLI1), and sonic hedgehog (SHH). Inhibition ofthe activity of an Hh signalling pathway target by an inhibitor may beless than 100%, e.g., about 10% to about 95%, e.g., 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or another percent inhibition of the activity of aHh signalling pathway target from about 10% to about 95%. An inhibitorof the Hh signalling pathway may be a small molecule (e.g., Sonidegib).Alternatively, the inhibitor may be a peptide, a protein, a nucleicacid, or a combination thereof, which can be administered directly inisolated form or indirectly via an expression vector (e.g., a plasmid orrecombinant virus for expression of the encoded inhibitor in a humansubject).

The term “agent that increases activation of a receptor of at least onetype II interferon and/or type I interferon,” as used herein, refers toany type of molecule that either directly or indirectly activate suchreceptors. Direct activators of such receptors include, but are notlimited to, administered exogenous interferons, administered exogenouspolynucleotides encoding interferons, administered peptides orpeptidomimetic agonists, and small molecule agonists capable ofactivating a receptor of at least one type II interferon and/or type Iinterferon. Indirect activators of such receptors refer to those theadministration of which result in increased production or secretion ofan endogenous interferon capable of activating a receptor of at leastone type II interferon and/or type I interferon. Such indirectactivators include, but are not limited to, checkpoint inhibitors orcombinations thereof that inhibit the activation of the PD-1 receptor,the CTLA-4 receptor, or both the PD-1 receptor and the CTLA-4 receptor.

The term “dosing period,” as used herein, refers to a defined period oftime over which at one or more doses of a first and a second therapeuticagent are administered to a subject in a desired temporal phaserelationship. For example, within a first dosing period, a smallmolecule inhibitor may be administered twice at least 10 days before thebeginning of administration of a recombinant virus expressing interferongamma. Thus, within the first dosing period the small molecule inhibitoris administered before the recombinant virus. If a second dosing periodcommences, and the same sequence of administration is continued, thesmall molecule inhibitor is still considered to be administered beforethe recombinant virus even though it occurs after the previousadministration of recombinant virus that occurred in the first dosingperiod.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an administered agent(e.g., a recombinant virus, an inhibitor the Hh signalling pathway, or apurified protein) which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. A“therapeutically effective amount” of a therapeutic agent that isadministered as part of a combination treatment can refer to an amountof the therapeutic agent that would be therapeutically effective whenused on its own (i.e., as a monotherapy), or may refer to a reducedamount that is therapeutically effective by virtue of its combinationwith one or more additional therapeutic agents.

The term “recombinant virus,” as used herein, refers to any virus thatis genetically modified by experimental intervention and is capable ofexpressing a polynucleotide from an experimentally introduced expressioncassette.

The term “administer” or “administered” as used herein, broadlyencompasses exposing a subject to a therapeutic agent in question bydirect or indirect means. In some cases, e.g., where a biotherapeuticagent such as a peptide, protein, or polynucleotide is to beadministered, it can be delivered directly as an isolated purifiedreagent by any suitable route of administration, or, alternatively, itcan be delivered indirectly by inducing expression of the biotherapeuticagent within the subject, e.g., by delivering to the human subject aplasmid DNA, modified mRNA, or a recombinant virus encoding the relevantbiotherapeutic agent.

The term “adverse event” as used herein, refers to any undesirableclinical occurrence in a subject/patient (as compared to the subject'sbaseline health) and is any untoward medical occurrence defined as anunintended disease or injury or untoward clinical signs (includingabnormal laboratory findings) in a patient. More specifically, grades ofadverse events, as referred to herein, include those published under the“Common Terminology Criteria for Adverse Events” published by the U.S.National Cancer Institute (version 4.03 published 14 Jun. 2010). Theseinclude mild (grade 1) adverse events which present as mild symptoms notrequiring medical intervention; moderate (grade 2) adverse events, whichrequire minimal, local or noninvasive intervention; severe or medicallysignificant (grade 3) adverse events, which are not immediatelylife-threatening, but may require hospitalization or prolongation ofhospitalization; life-threatening (grade 4) adverse events requiringurgent intervention; and adverse event-related death (grade 5). Adverseevents commonly associated with administration of agents for inhibitingthe Hh signalling pathway for cancer therapy include, but are notlimited to, myopathies, fatigue, alopecia (hair loss), dysgeusia(distortion of taste perception), weight loss, elevation of creatinephosphokinase, muscle cramps/spasms, and ovarian dysfunction, and newonset squamous cell carcinoma.

The terms “encoding” “encodes” “encoded” and the like as used herein,refer to any biomolecule for which a corresponding DNA or RNA sequencecan be either transcribed into one or more RNAs or translated into oneor more peptides or proteins. Examples of such encodable biomoleculesinclude, but are not limited to, miRNAs, siRNAs, antisense RNAs, gRNAs,proteins, and peptides.

The term “expressing” or “expression” as used herein refers to theprocess of transcription and/or translation.

The term “purified” as used herein, in relation to a protein (e.g.,“purified interferon gamma” and the like) refers to a protein providedin a form that is substantially free of contaminants normally associatedwith the protein in a native or natural environment.

The term “antibody” as used herein, includes polyclonal antibodies,monoclonal antibodies, bispecific antibodies, fusion diabodies,triabodies, heteroconjugate antibodies, chimeric antibodies includingintact molecules as well as fragments thereof, and other antibody-likemolecules. Antibodies include modifications in a variety of formsincluding, for example, but not limited to, domain antibodies includingeither the VH or VL domain, a dimer of the heavy chain variable region(VHH, as described for a camelid), a dimer of the light chain variableregion (VLL), Fv fragments containing only the light (VL) and heavychain (VH) variable regions which may be joined directly or through alinker, or Fd fragments containing the heavy chain variable region andthe CH1 domain.

The term “antigen-binding portion” as used herein, refers to a region onan antibody that binds to a specific antigen. Typically, such anantigen-binding portion will include at least a heavy chain variabledomain (V_(L)) and a light chain variable domain (V_(H)), which togetherform the antigen-binding portion. An example of such an antigen-bindingportion would include, e.g., single chain variable fragments (scFvs).

The term “small molecule” as used herein, refers to a chemical compoundsor molecule having a molecular weight below 2000 daltons.

The terms “synergy” or “synergistic” as used herein refer to an effect(e.g., induction of cell death”) resulting from the use of a combinationof agents where the effect is quantitatively greater than the sum of theeffects resulting from the use of each agent separately. For example, ifagent “A” causes 30% cell death and agent “B” causes 30% cell death, the(non-synergistic) sum of such effects would be 60%. If, in fact, thecombination of agents A and B results in greater than 60% cell death,their combined effect would be considered synergistic.

The terms “treating” or “treatment” as used herein, refer to both directtreatment of a subject by a medical professional (e.g., by administeringa therapeutic agent to the subject), or indirect treatment, effected, byat least one party, (e.g., a medical doctor, a nurse, a pharmacist, or apharmaceutical sales representative) by providing instructions, in anyform, that (i) instruct a subject to self-treat according to a claimedmethod (e.g., self-administer a drug) or (ii) instruct a third party totreat a subject according to a claimed method. Also encompassed withinthe meaning of the term “treating” or “treatment” are prevention ofrelapse or reduction of the disease to be treated, e.g., byadministering a therapeutic at a sufficiently early phase of disease toprevent or slow its progression.

Treatment of a Disease Characterised by Aberrant Cell Proliferation

The methods described herein relate to treating a human subjectsuffering from a disease characterised by aberrant cell proliferation,by administering a combination regimen of: (i) at least one agent thatincreases activation of a receptor of at least one type II interferonand/or type I interferon; and (ii) at least one agent that inhibits theHedgehog (Hh) signalling pathway. Whilst not wishing to be bound bytheory, inhibition of Hh pathway signalling in conjunction withincreased type II and/or type I interferon receptor activity results ina synergistic anti-proliferative effect that will reduce treatmentdosing and/or duration as compared to monotherapy.

In some embodiments the treatment methods described herein are performedon a mammalian subject such as a cow, sheep, horse, cat, mouse, rat,guinea pig, dog, pig, non-human primate, or a human. In some preferredembodiments the subject to be treated is a human subject.

Cancers that can be treated by any of the methods provided hereininclude, but are not limited, to basal cell carcinoma, melanoma,lymphoma, squamous cell carcinoma, Merkel cell carcinoma, lung cancer,prostate cancer, sarcomas, medulloblastomas, cancers with desmoplasticstromas, colorectal cancer, ovarian cancer, breast cancer, gastriccancer, pancreatic cancer, mesothelioma, mesenchymal cancer, epithelialcancer, and adenocarcinomas. In some preferred embodiments, the subjectto be treated is suffering from basal cell carcinoma. In someembodiments, the subject suffering from basal cell carcinoma (BCC)suffers from Basal Cell Nevus Syndrome (BCNS) or sporadic BCC. In someembodiments the treatment methods are performed on a subject sufferingfrom a recurrent cancer or relapsing cancer.

Fibrotic diseases that can be treated by the methods provided hereininclude, but are not limited to, Keloids, Scleroderma/systemicsclerosis, Nephrogenic systemic fibrosis, adhesive capsulitis,Dupuytren's contracture, and Arthrofibrosis.

In preferred embodiments, the dose of an agent that inhibits theHedgehog (Hh) signalling pathway (e.g., Visomodegib), the dose of a(type II or type I) interferon (or agonist or checkpoint inhibitor), orboth in a combination treatment may be reduced relative to a standarddose accepted in the art for administration of each agent alone. In someembodiments, of the treatment methods described herein a reduced doseavoids induction of at least one adverse event in the subject beingtreated. In some preferred embodiments, the dose to be administered isreduced by about 25% to about 95% relative to a standard dose, e.g., a30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or another percentdose reduction relative to a standard dose for the aberrant cellproliferation disease to be treated in a range of about 25% to about95%. In some preferred embodiments, the dose is reduced by about 50% toabout 85% relative to the a standard dose.

Symptoms, diagnostic tests, and prognostic tests for various typesdiseases characterised by aberrant cell proliferation are known in theart. See, e.g., the website of the National Comprehensive CancerNetwork: (nccn.org/professionals/physician_gls/f_guidelines.asp), andBritish Medical Journal (BMJ) Best Practice (website at:bestpractice.bmj.com), respectively.

Inhibition of the Hedgehog Signalling Pathway

In some embodiments an agent that inhibits the Hh signalling pathwayinhibits one or more Hh signalling pathway targets selected from thegroup consisting of: SMO, PTCH1, GLI, SHHat, tGLI1, SHH, and acombination thereof. The inhibitors to be administered can include, butare not limited to, small molecules, peptides, proteins, nucleic acids,or a combination thereof.

In some embodiments the agent is a small molecule inhibitor of the Hhsignalling pathway. Suitable small molecule inhibitors of the Hhsignalling pathway include, but are not limited to, Sonidegib/LDE225(CAS 956697-53-3), Saridegib (CAS 1037210-93-7), Vismodegib (CAS879085-55-9), LEQ-506 (CAS 1204975-42-7), Taledegib (CAS 1258861-20-9),Itraconazole (CAS 84625-61-6), Glasdegib (CAS 1095173-27-5), GANT61 (CAS500579-04-4), Jervine (CAS 469-59-0), CUR61414 (CAS 334998-36-6),BMS-833923 (CAS 1059734-66-5), TAK-441 (CAS 1186231-83-3), MRT-92 (CAS1428307-52-1), HH-13 (Li et al., 2019), GANT61 (CAS 500579-04-4), andHH-20 (Li et al., 2019). In some preferred embodiments the smallmolecule inhibitor to be administered is Vismodegib.

In some embodiments the agent that inhibits the Hh signalling pathway isa polypeptide. In some embodiments the polypeptide comprises an antibodyor an antigen-binding portion thereof that binds specifically to an Hhsignalling pathway target. In some embodiments the polypeptide comprisesan antibody or an antigen-binding portion thereof against SHH, SMO,PTCH1, GLI, SHHat, or tGLI1. In some embodiments the polypeptidecomprises an antibody or an antigen-binding portion thereof against SHH.Suitable examples of antibodies against SHH include, but are not limitedto, 5E1, MEDI-5304, and antibodies that compete with any one of 5E1,MEDI 5304, or 1C11-2G4 for binding to SHH.

Suitable examples of antibodies against other Hh signalling pathwaytargets include, but are not limited to, α-Ptch1 against PTCH1 (Nakamuraet al., 2007) and E5 against SMO (Santa Cruz Biotechnology, Cat No.sc-166685).

In some embodiments a polypeptide agent for inhibiting the Hh signallingpathway is a (non-antibody) negative regulator. In some embodiments thenegative regulator is Rab 23 (Chi et al., 2012), human suppressor offused hSu(fu) (Stone et al., 1999, or dominant negative SHH-G27A (Singhet al., 2009). In some embodiments the protein or peptide inhibitors areprovided as conjugates or fusion proteins that include a cellpenetrating peptide (CPP). CPPs are known in the art and commerciallyavailable (see, for example, Hoffmann et al., 2018). In someembodiments, where the inhibitor is a protein, the protein is anantibody inhibitor of the Hh signalling pathway. In some embodiments theinhibitor of the Hh signalling pathway includes a peptide. In someembodiments a peptide inhibitor comprises the amino acid sequence ofHL2-m5 corresponding to SEQ ID NO:1:

where O2beY corresponds to the unnatural amino acidO-(2-bromoethyl)-tyrosine (Owens et al., 2017).

In other embodiments the the agent that inhibits the Hh signallingpathway is a polynucleotide. In some embodiments the polynucleotideencodes any of the above-described polypeptides or peptides forinhibiting the Hh signalling pathway (e.g., an antibody against SHH).

In other embodiments, the polynucleotide acid does not encode apolypeptide that inhibits the Hh signalling pathway, but rather is orencodes a miRNA, an siRNA, an RNAi, or a CRISPR gRNA, an antisense RNA,or an antisense oligonucleotide targeted against a Hh signalling pathwaytarget. The skilled person will appreciate that where a polynucleotideis utilised for expression of an RNA or protein, a suitable vector forexpression of the RNA and/or protein in a mammalian subject can beadministered to the subject to be treated either by in vivo transfectionor by viral transduction using a recombinant virus as described herein.

In other embodiments the polynucleotide is an miRNA. Suitable miRNAsinclude, but are not limited to, miR-125b, miR-323, miR324-5p, miR-210,and miR-14. In other embodiments, the polynucleotide is an antisenseRNA. Suitable antisense RNA inhibitors of the Hh signalling pathwayinclude, but are not limited to, antisense oligomers against GLI1 (Hegdeet al., 2012), GLI2 (Narita et al., 2008), and SMO (Gao et al., 2006).In other embodiments the polynucleotide to be administered comprises agRNA for CRISPR targeting of the Hh signalling pathway. Suitableexamples of gRNAs include, but are not limited to, gRNAs against GLI1(Diao et al., 2018), GLI2 (Hsiao et al., 2018), and SMO (Wu et al.,2018). It will be appreciated by those skilled in the art that targetinggRNAs can be used for knock out or knock down of a target gene(Konermann et al., 2018) depending on the targeting enzyme (e.g., Cas9)used in conjunction with the gRNA.

In some embodiments, where the polynucleotide is to be administered bytransient in vivo transfection, the polynucleotide is a chemicallymodified (coding or non-coding) RNA in which a proportion (e.g., 10%,30%, 50%, or 100%) of at least one type of nucleotide, e.g., cytosine,is chemically modified to increase its stability in vivo. For example,in some cases modified cystosines are 5-methylcytosines. Suchpolynucleotides are particularly useful for delivery/transfection tocells in vivo, especially when combined with a transfection/deliveryagent. In some cases, a chemically modified RNA is a chemically modifiedRNA in which a majority of (e.g., all) cystosines are 5-methylcytosines,and where a majority (e.g., all) of uracils are pseudouracils. Thesynthesis and use of such modified RNAs are described in, e.g., WO2011/130624. Methods for in vivo transfection of DNA and RNApolynucleotides are known in the art as summarised in, e.g., Liu et al.(2015) and Youn et al. (2015).

Agents for Increasing Type II and/or Type I Interferon Receptor Activity

In some embodiments an agent to be administered for increasing Type IIand/or Type I interferon receptor activity includes an exogenousinterferon. In some embodiments of the treatment methods describedherein, the amino acid sequence of the interferon to be administeredcomprises an amino acid sequence at least about 80% identical to theamino acid sequence of a human type II interferon, e.g., humaninterferon gamma (GenBank No. NP_000610.2), e.g., 82%, 85%, 88%, 90%,92%, 95%, 97%, 99%, or another percent identical to the human interferongamma sequence ranging from about 80% to 100% identical to the humaninterferon gamma sequence. In preferred embodiments the amino acidsequence of the interferon gamma to be administered is 100% identical tothe amino acid sequence of human interferon gamma. In other embodimentsthe interferon gamma to be administered comprises an amino acid sequenceat least about 80% identical to the amino acid sequence of a human typeI human interferon, e.g., 82%, 85%, 88%, 90%, 92%, 95%, 97%, 99%, oranother percent identical to the amino acid sequence of a human type Iinterferon. Suitable human type I interferons include one or more of:interferon alpha (GenBank No. AAA52724.1), interferon beta (GenBank No.AAC41702.1), interferon epsilon (GenBank No. AAQ88933), interferon kappa(GenBank No. EAW58563.1), and interferon omega 1 (GenBank No.CAA41626.1).

In some embodiments interferon gamma is administered as a substantiallypurified protein at level of purity considered to be safe for humanadministration, e.g., generally greater than 99% purity and about 5×10⁶IU/mg to about 4×10⁷ IU/mg.

In other embodiments the type II or type I interferon to be administeredis administered indirectly as a polynucleotide encoding a type II ortype I interferon to be expressed in a human subject. In some preferredembodiments, the amino acid sequence of the encoded interferon is a typeII interferon at least about 80% identical to the amino acid sequence ofhuman interferon gamma (GenBank No. NP_000610.2), e.g., 82%, 85%, 88%,90%, 92%, 95%, 97%, 99%, or another percent identical to the humaninterferon gamma sequence ranging from about 80% to 100% identical tothe human interferon gamma sequence. In preferred embodiments the aminoacid sequence of the encoded interferon gamma is 100% identical to theamino acid sequence of human interferon gamma.

In some preferred embodiments, where the type II or type I interferon isto be administered as a polynucleotide encoding the type II or type Iinterferon, the polynucleotide is administered via a recombinant virusfor expression in a subject to be treated. In some embodiments, whererecombinant virus is used for expression of the type II or type Iinterferon in a human subject, the same recombinant virus co-expressesan inhibitor of the Hh signalling pathway as described herein.

In some embodiments, where a type II interferon, a type I interferon isto be administered in the form of a polynucleotide as described herein,the polynucleotide is administered by transient in vivo transfection ofa “naked” polynucleotide (e.g., a plasmid expression vector or anexpression amplicon).

In some embodiments an agent for increasing type II and/or type Iinterferon receptor activity is an agent that stimulates endogenousproduction and/or release of a type II interferon and/or type Iinterferon in the subject. In some preferred embodiments the agentstimulates endogenous production and/or release of interferon gamma inthe subject. In some embodiments the agent for increasing type II and/ortype I interferon receptor activity includes one or more checkpointinhibitors. In some embodiments the one or more checkpoint inhibitorsinhibit activation of the PD-1 receptor, CTLA-receptor, or both. In someembodiments the agent comprises one or more checkpoint inhibitors thatinhibit activation of the PD-1 receptor and inhibit activation of theCTLA-4 receptor.

In some embodiments the one or more checkpoint inhibitors include apolypeptide or a peptide. In some embodiments the checkpoint inhibitorpolypeptide or the checkpoint inhibitor peptide bind to a checkpointreceptor. In other embodiments the checkpoint inhibitor polypeptide orthe checkpoint inhibitor peptide bind to a checkpoint receptor ligand.In some embodiments, where the checkpoint inhibitor polypeptide or thecheckpoint inhibitor peptide bind to a checkpoint receptor ligand, thecheckpoint receptor ligand includes PD-L1, PD-L2, CD80/B7-1, CD86/B7-2,or any combination thereof.

In some embodiments, where the one or more checkpoint inhibitors includea polypeptide, the polypeptide is an antibody or antigen-binding portionthereof. Suitable checkpoint inhibitor antibodies include, but are notlimited to, Ipilimumab, Pembrolizumab, Nivolumab, Atezolizumab,Avelumab, Durvalumab, Cemiplimab, AGEN1181, Tremelimumab, and anycombination thereof. In some embodiments, where the one or morecheckpoint inhibitors includes an antibody, the antibody is bispecific.Suitable bispecific antibodies include, but are not limited to, M7824(see Clinical Trial Identifier NCT02699515), MGD013 (NCT03219268),FS118, MCLA-134, XmAb-20717, and ATOR-1015 (see, e.g., Dahlen et al.,2018).

In some embodiments the one or more checkpoint inhibitors include apeptide. Examples of suitable peptide checkpoint inhibitors include, butare not limited to, (D)PPA-1 (Chang et al., 2015), PL120131 (Boohaker etal., 2018), TPP-1 (Li et al., 2018), UNP-12 (Sasikumar et al., 2013),NP-12 (Saskikumar et al., 2019), and any combination thereof.

In other embodiments the one or more checkpoint inhibitors include atargeting polynucleotide. Suitable examples of targeting polynucleotidesinclude siRNA, RNAi, antisense oligonucleotides, and any combinationthereof. Such targeting polynucleotides may be directed againstexpression of one or more of, e.g., PD-1, PD-L1, PD-L2, CTLA4,CD80/B7-1, CD86/B7-2, or any combination thereof. Suitable targetingpolynucleotides are known in the art, e.g., MN-siPDL1 (Yoo et al., 2019)and gRNA against PD-1 Choi et al. (2019).

Recombinant Viruses

In some embodiments, where (i) the at least one agent that inhibits theHh signalling pathway and/or (ii) the at least one agent (e.g., acheckpoint inhibitor) that increases activation of a receptor of atleast one type II interferon and/or type I interferon comprises apeptide, a polypeptide, or a polynucleotide, a recombinant virus isadministered to induce expression of the peptide, polypeptide, orpolynucleotide when administered to the subject (e.g., a human subject).A variety of recombinant virus types are suitable for expression of atype II or type I interferon and, optionally, a polypeptide, apolynucleotide, or a peptide that inhibits the Hh signalling pathway asdescribed herein.

In some embodiments, the recombinant virus to be administered is a DNAvirus. Suitable types of DNA viruses include adenovirus,adeno-associated virus (AAV), herpes simplex virus (HSV), retrovirus,and lentivirus. Methods for design, production, and use of such types ofrecombinant DNA viruses are established in the art, as exemplified inFukazawa et al. (2010) and in “Gene Therapy Protocols” for adenovirus;“Adeno-Associated Virus: Methods and Protocols” for AAV; Cody et al.(2013) and “Herpes Simplex Virus: Methods and Protocols” for HSV; “GeneTherapy Protocols Vol. 1: Production and In Vivo Applications of GeneTransfer Vectors” and Amer et al. (2014) for retrovirus; and Merten etal. (2016) and Emeagi et al. (2013) for lentivirus. In some preferredembodiments, the recombinant virus to be used in the treatment method isan adenovirus. In some preferred embodiments the recombinant adenovirusis ASN-002, a replication-deficient type 5 adenovirus for expression ofinterferon gamma (also known as Tg1042 and SP-002) (Urosevic, 2007; Liuet al., 2004; Dummer et al., 2004 and 2010; Accart et al., 2013;Khammari et al., 2015; Dreno et al., 2014; Hillman et al., 2004).

In other embodiments, the recombinant virus to be administered is arecombinant, replication-deficient or replication-competent RNA virus.Suitable types of replication deficient or replication-competent RNAviruses Alphavirus (e.g., Sindbis or Semliki Forest Virus), Flavivirus(e.g., Kunjin virus), Paramyxovirus (e.g., Sendai virus), Rhabdovirus(e.g., vesicular stomatitis virus), and Orthomyxovirus (e.g., influenzaA virus). Methods for design, production, and use of such types ofrecombinant RNA viruses are established in the art, as exemplified inLundstrom (2015) and Quetglas et al. (2010) for Alphavirus; Hoang-Le etal. (2009) and Usme-Ciro et al. (2013) for Flavivirus; Cattaneo (2010)for Paramyxovirus; Finke et al. (2005) and Chang et al. (2010) forRhabdovirus; and U.S. Pat. No. 8,475,806 for Orthomyxovirus.

In some embodiments, the recombinant DNA or RNA virus is a replicationdeficient virus incapable of replication in transduced cells. In otherembodiments, the recombinant virus is a replication-competent virus,which can replicate in a transduced host cell. Alternatively, therecombinant virus is a conditionally replication-competent virus thatcan replicate only in particular cell types or in cells with aparticular expression profile, e.g., p53-deficient cancer cells.

Examples of suitable promoters for driving expression of biotherapeuticagents from a recombinant virus in a method described herein include,but are not limited to, constitutive promoters such as, CMV, CAG,EF-1-I, HSV1-TK, SV40,

-actin, and PGK promoters. In other embodiments, a promoter is aninducible promoters, such as those containing TET-operator elements. Incertain embodiments, target-selective promoters are used to driveexpression of biotherapeutic agents in specific cell types orspecifically in cells exhibiting aberrant cell proliferation. Examplesof suitable promoters useful for the methods described herein include,but are not limited to, the erb 2 promoter (breast cancer), thecarcinoembryonic antigen promoter (colorectal cancer), theurokinase-type plasminogen activator receptor promoter (colorectalcancer), the tyrosinase promoter (melanoma), the melacortin receptor(melanoma); the human telomerase reverse transcriptase (hTERT) promoter(multiple cancers), the RAS-related nuclear protein promoter (multiplecancers), the breast cancer metastasis suppressor 1 promoter (multiplecancers), the Rad51C promoter (multiple cancers), and the minichromosomemaintenance complex component 5 promoter (multiple cancers).

In some embodiments, where two or more proteins are to be expressed froma recombinant virus, the recombinant virus contains an expressioncassette encoding a polycistronic mRNA (a “polycistronic expressioncassette”), which, upon translation gives rise to independentpolypeptides comprising different amino acid sequences orfunctionalities, e.g., interferon gamma and a protein inhibitor of theHh signalling pathway (e.g., hSu(fu)). In some embodiments, apolycistronic expression cassette encodes a “polyprotein” comprisingmultiple polypeptide sequences that are separated by encoded by apicornavirus, e.g., a foot-and-mouth disease virus (FMDV) viral 2Apeptide sequence. The 2A peptide sequence acts co-translationally, bypreventing the formation of a normal peptide bond between the conservedglycine and last proline, resulting in ribosome skipping to the nextcodon, and the nascent peptide cleaving between the Gly and Pro. Aftercleavage, the short 2A peptide remains fused to the C-terminus of the‘upstream’ protein, while the proline is added to the N-terminus of the‘downstream’ protein. which during translation allow cleavage of thenascent polypeptide sequence into separate polypeptides. See, e.g.,Trichas et al. (2008).

In other embodiments, a polycistronic expression cassette mayincorporate one or more internal ribosomal entry site (IRES) sequencesbetween open reading frames incorporated into the polycistronicexpression cassette. IRES sequences and their use are known in the artas exemplified in, e.g., Martinez-Sales (1999).

In some embodiments, a recombinant virus used in the method has targetedtropism, e.g., tropism for a particular cell type as reviewed in Bucholzet al. (2015). Suitable targeting moieties, to be incorporated into arecombinant viral capsid surface, include ligands that bind to cellsurface receptors that are overexpressed by overproliferating cells(e.g., cancer cells). For example, the Her2/neu receptor, frequentlyoverexpressed in breast cancer cells, can be targeted by incorporating adesigned ankryrin repeat protein (DARPin) ligand, as has been done forlentivirus (Münch et al. 2011) in AAV (Münch et al. 2013). In anotherexample a recombinant lentivirus is designed to target P-glycoprotein,overexpressed on the surface of melanoma cells, by incorporating anantibody into the viral capsid surface (Morizono et al. 2005).

Dosing Regimens

The person of ordinary skill in the art will appreciate that a suitabletherapeutically effective dose of an agent that inhibits the Hhsignalling pathway when administered in combination with an agent thatincreases activation of a receptor of at least one type II interferonand/or type I interferon, as described herein, will depend upon factorssuch as the particular Hh signalling pathway inhibitor, the developmentof adverse effects of the treatment, the particular agent, the diseasestage, the characteristics of the subject or host in need of treatment(e.g., weight), the properties of the particular type of disease to betreated, the proposed route of administration, etc., but cannevertheless be determined in a manner known in the art. The desireddose may conveniently be presented in a single dose or as divided dosesadministered simultaneously (or over a short period of time) or atappropriate intervals, for example as two, three, four or more sub-dosesper day.

In general, agents being administered in combination do not necessarilyhave to be administered in the same pharmaceutical composition, and may,because of different physical and chemical characteristics, beadministered by different routes.

The time period between the multiple administration steps may rangefrom, a few minutes to several days, depending upon the properties ofeach pharmaceutical agent, such as potency, solubility, bioavailability,plasma half-life and kinetic profile of the pharmaceutical agent.Circadian variation of various physiological parameters may also beevaluated to determine the optimal dose interval.

Initial administration can be via any route practical, such as, forexample, an intravenous injection, a bolus injection, infusion over 5minutes to about 5 hours, a pill, a capsule, topical application,transdermal patch, and the like, or combination thereof. In someembodiments, administration is by sub-cutaneously, such as intralesionalinjection perilesional injection, or injection in close proximity to alesion (for example with about 5 cm or within about 1 cm). In someembodiments a Hh signalling pathway inhibitor is administered orally,and the type II or type I interferon (e.g., by way of a recombinantvirus) is administered intralesionally.

In some embodiments of the treatment methods described herein either of:(i) at least one agent that increases activation of a receptor of atleast one type II interferon and/or type I interferon; or (ii) at leastone agent that inhibits the Hedgehog (Hh) signalling pathway isadministered systemically, intralesionally, or topically.

In some preferred embodiments, e.g., where the subject to be treatedpresents with lesions/tumours amenable to direct local administration(e.g., basal cell carcinoma), administration of (i) is intralesional. Insome embodiments, administration of (i) is intralesional andadministration of (ii) is systemic.

In some embodiments administration of each of (i) and (ii) is performedseparately during a first dosing period, i.e., a period over which afirst set of doses of (i) and (ii) are administered to the subject. Insome embodiments, during the first dosing period, (ii) is administeredafter beginning dosing with (i). In some embodiments (ii) isadministered at least one day to about eight weeks after beginningadministration of (i), e.g., one day, two days, three days, four days,five days, six days, one week, two weeks, three weeks, four weeks, sixweeks, seven weeks, or another period from at least one day to abouteight weeks after beginning administration of (i). In some embodiments(ii) is administered at least one to three weeks after beginningadministration of (i).

In other embodiments, during the first dosing period (ii) isadministered before administration of (i). In some embodiments (ii) isadministered at least one day to about eight weeks before beginningadministration of (i), e.g., one day, two days, three days, four days,five days, six days, one week, two weeks, three weeks, four weeks, sixweeks, seven weeks, or another period from at least one day to abouteight weeks before beginning administration of (i). In some preferredembodiments (ii) is administered about 10 days to about 14 days beforebeginning administration of (i).

In some embodiments (i) and (ii) are co-administered during a firstdosing period, i.e., they are administered to the subject within lessthan one day of each other. In some embodiments co-administration of (i)and (ii) includes administration of (i) and (ii) within about 1 minuteto about 22 hours of each other within a first dosing period, e.g., 5minutes, 30 minutes, 1 hours, 2 hours, 8 hours, 12 hours, 18, hours oranother time interval from about 1 minute to about 22 hours during afirst dosing period. In some embodiments, where (i) and (ii) areco-administered, they are co-administered in a single formulationcontaining both (i) and (ii).

In some embodiments (i), (ii), or both (i) and (ii) are administeredmultiple times during a first dosing period. In some embodiments (i),(ii), or both (i) and (ii) are administered at a frequency of betweenabout once per week to about once per day during a first dosing period,e.g., once per five days, once per four days, once per three days, onceper two days, or another frequency from about once per week to aboutonce per day. In some embodiments, where (i) and (ii) are administeredmultiple times during a first dosing period, the dosing frequency for(ii) is lower than that for (i). In other embodiments the dosingfrequency for (i) is lower than that for (ii). In some embodiments thedosing frequency for (i) and (ii) is the same.

In some embodiments, a subject to be treated is administered acombination treatment as described herein over multiple dosing periodsincluding at least first and second dosing periods. The number of dosingperiods may range from 1 to 14, e.g., 2, 3, 4, 5, 6, 8, 10, 12, oranother number of dosing periods from 1 to 14. In some embodiments thetreatment includes at least first and second dosing periods. Where asubject is treated over multiple dosing periods, the total aggregatedose (i) and/or (ii) may be varied among different dosing periods.

Monotherapy for cell proliferation diseases (e.g., cancer) with agentsthat inhibit the Hh signalling pathway for treatment of a disease aregenerally acknowledged to cause adverse events especially in view of theduration (usually about 10 months) and dose of these agents required foran effective response. However, such adverse effects (e.g., musclecramps/spasms, fatigue, and myopathies) commonly result in patientsstopping treatment with such agents before a full course of treatmenthas been completed. Indeed, adverse events (e.g., muscle spasms) oftenbegin as early as two months into the treatment (Lacouture et al, 2018).In contrast, in the combination treatment methods described herein,synergy between treatment with (i) at least one agent that increasesactivation of a receptor of at least one type II interferon and/or typeI interferon; and (ii) agent that inhibits the Hedgehog (Hh) signallingpathway affords a substantially reduced duration and/or dose of (ii).Thus, in some embodiments the dose of (ii) administered or treatmentperiod with (ii) in combination with (i) is limited to avoid inductionof at least one adverse event associated with treatment with (ii) alone.Accordingly, in some embodiments the dose of (ii) administered in acombination treatment with (i) as described herein is about 10% to about90% less than a dose of (ii) required for monotherapy with (ii) while atthe same time avoiding induction of more than a moderate eventassociated with monotherapy treatment with (ii), e.g., 20% less, 30%less, 40%, less, 50% less, 60% less, 70% less, 80% less, or anothervalue from about 10% to about 90% less than a dose of (ii) required formonotherapy with (ii) while avoiding induction of more than a moderateevent associated with monotherapy treatment with (ii). In otherembodiments the duration of the treatment period with (ii) administeredin a combination treatment with (i) as described herein is about 10% toabout 90% less time than the duration of the treatment with (ii)required for monotherapy with (ii) while at the same time avoidinginduction of more than a moderate event associated with monotherapytreatment with (ii), e.g., 20% less, 30% less, 40%, less, 50% less, 60%less, 70% less, 80% less, or another value from about 10% to about 90%less time than the duration of the treatment period with (ii) requiredfor monotherapy with (ii) while avoiding induction of more than amoderate event associated with monotherapy treatment with (ii).

In some embodiments, where the subject to be treated is suffering frombasal cell carcinoma, the a dosing period comprises 2-3 administrationsof (i) in a single week and daily administration of (ii). In otherembodiments, where the subject to be treated is suffering from basalcell carcinoma, a dosing period comprise 2-3 administrations in twoweeks.

In some embodiments a treatment duration, which includes all dosingperiods, is from about 3 weeks to about 40 weeks, e.g., 4 weeks, 5weeks, 8 weeks, 12 weeks, 16 weeks, 18 weeks, 20 weeks, 24 weeks, 30weeks, 32 weeks, 34 weeks, 36 weeks, or another treatment duration fromabout 3 weeks to about 40 weeks.

In some embodiments, administration of a recombinant virus forexpression of a type II and/or type I interferon and, optionally,expression of an inhibitor of the Hh signalling pathway inhibitor, is byan intralesional route of administration. In some embodiments, where apolypeptide (e.g., an antibody) or polynucleotide (e.g., shRNA) toinhibit the Hh signalling pathway is to be expressed from a recombinantvirus, it is co-expressed with a type II or type I interferon from thesame recombinant virus. In other embodiments, where a polypeptide (e.g.,an antibody) or polynucleotide (e.g., shRNA) to inhibit the Hhsignalling pathway is to be expressed in a subject by administering arecombinant virus, it is expressed from a recombinant virus that isseparate and distinct from the recombinant expression virus used todrive expression of a type II or type I interferon in a human subject.

In some embodiments, the administered intralesional total dose ofrecombinant virus is from about 1×10⁷ viral particles/lesion to about1×10¹² viral particles/lesion, e.g., 2×10⁷, 3×10⁷, 4×10⁷, 5×10⁷, 6×10⁷,8×10⁷, 1×10⁸, 1.5×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 6×10⁸, 8×10⁸, 9×10⁸, 1×10⁹,2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 8×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰,4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 8×10¹¹, 9×10¹¹, or another number of viralparticles/lesion from about 1×10⁷ viral particles/lesion to about 1×10¹²viral particles/lesion. In some embodiments, the intralesional viraldose ranges from about 2×10¹⁰ viral particles/lesion to about 3×10¹¹viral particles/lesion. In some embodiments the recombinant virus forexpression of the therapeutically effective amount of the type IIinterferon and/or a type I interferon is administered as at least 5×10⁷viral particles to about 5×10⁹ viral particles per lesion per dosingday. In some preferred embodiments the recombinant virus for expressionof the therapeutically effective amount of the type II interferon and/ora type I interferon is administered as at least 1×10⁸ viral particles toabout 1×10⁹ viral particles per lesion per dosing day. In some preferredembodiments the recombinant virus for expression of the therapeuticallyeffective amount of the type II interferon and/or a type I interferon isadministered as at least 1×10⁹ viral particles per lesion per dosingday.

In some embodiments, the subject to be treated is administered multipledoses of a recombinant virus expressing a Type I or Type II interferonin a combination treatment with a small molecule inhibitor of the Hhsignalling pathway in each dosing period.

In other embodiments, where administration of the recombinant virus isintralesional, the total aggregate dose of recombinant viral particlesper dosing period ranges from about 1×10⁸ viral particles/lesion toabout 1×10¹³ viral particles/lesion, e.g., 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 8×10⁸, 1×10⁹, 1.5×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 6×10⁹, 8×10⁹, 9×10⁹,1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 8×10¹⁰, 1×10¹¹, 2×10¹¹,3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.5×10¹²,2×10¹², 3×10¹², 4×10¹², 5×10¹², 6×10¹², 8×10¹², 9×10¹² or another numberof total viral particles per dosing period from about 1×10⁸ viralparticles/lesion to about 1×10¹³ viral particles/lesion.

In some embodiments, where administration of the recombinant virus is bysystemic, intraperitoneal, or intrapleural administration, the totalaggregate viral dose per dosing period for a recombinant virus is about1×10⁹ viral particles to about 1×10¹⁴ viral particles per dosing period,e.g., 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 8×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰,4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 1.5×10¹¹, 2×10¹¹,3×10¹¹, 4×10¹¹, 6×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹²,5×10¹², 6×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 8×10¹³, 9×10¹³ or another number of total viral particles perdosing period from about 1×10⁹ viral particles to about 1×10¹⁴ viralparticles.

In some embodiments, where the agent that inhibits the Hh signallingpathway is a small molecule inhibitor (e.g., Vismodegib), andadministration is intralesional, the dose concentration is at leastabout 5 μM to about 40 μM per lesion, e.g., 10 μM, 15 μM, 20 μM, 25 μM,30 μM, 35 μM per lesion, or another dose concentration from about 5 μMto about 40 μM. In some embodiments, the dose concentration is at leastabout 20 μM to about 100 μM per lesion, e.g., 25 μM, 30 μM, 45 μM, 50μM, 60 μM, 70 μM, 80 μM, 90 μM, or another concentration per lesion fromabout 20 μM to about 100 μM. In other embodiments the intralesional doseof the small molecule inhibitor (e.g., Vismodegib) ranges from about0.02 mg/lesion to about 10 mg/lesion per administration, e.g., about0.05 mg/lesion, 0.075 mg/lesion, 0.1 mg/lesion, 0.15 mg/lesion, 0.2mg/lesion, 0.25 mg/lesion, 0.3 mg/lesion, 0.5 mg/lesion, 0.7 mg/lesion,0.8 mg/lesion, 1 mg/lesion, 1.5 mg/lesion, 2 mg/lesion, 3 mg/lesion, 3.5mg/lesion, 4 mg/lesion, 4.5 mg/lesion, 5 mg/lesion, 5.5 mg/lesion, 6mg/lesion, 7 mg/lesion, 8 mg/lesion, 9 mg/lesion or anotherintralesional dose from about 0.02 mg/lesion to about 10 mg/lesion peradministration. In some preferred embodiments, the intralesional dose ofa small molecule Hh signalling pathway inhibitor ranges from about 2.5mg/lesion to about 10 mg/lesion.

In some embodiments, a disease to be treated, as described herein,includes treatment by systemic administration of one or more inhibitorsas described in the combination treatment methods provided herein. Inother embodiments, the administration is intraperitoneal administration.In some embodiments, the administration is intrapleural administration.

In some embodiments, where the agent that inhibits the Hh signallingpathway is a small molecule inhibitor (e.g., Vismodegib), andadministration is systemic, the dose concentration in circulation isabout 10 μM to about 80 μM, e.g., 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40μM, 45 μM, 50 μM, 60 μM, 70 μM, or another concentration in circulationfrom about 10 μM to about 80 μM. In some preferred embodiments, the doseconcentration in circulation is at least about 20 μM to about 40 μM,e.g., 25 μM, 30 μM, 35 μM, or another concentration in circulation fromat least about 20 μM to about 40 μM. In some embodiments, where the oneor more inhibitors is administered by systemic, intraperitoneal, orintrapleural administration, each administered dose is in a range ofabout 0.1 mg/kg to about 12 mg/kg per administration, e.g., 0.20 mg/kg,0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4mg/kg, 4.5 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 10 mg/kg, oranother dose ranging from about 0.1 mg/kg to about 12 mg/kg. In somepreferred embodiments, the dose of the one or more inhibitors is about 2mg/kg to about 5 mg/kg.

In some embodiments the small molecule inhibitor of the Hh signallingpathway is administered at a dose of about 50 mg to about 500 mg perdosing day, e.g., 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 450 mg,or another total dose of about 50 mg to about 500 mg per dosing day. Insome embodiments the small molecule inhibitor of the Hh signallingpathway is administered at a total dose of about 150 mg per dosing day.

In other embodiments, treatment of a disease, as described herein (e.g.,a skin cancer), includes topical administration of an inhibitor asdescried herein. In some embodiments, an inhibitor is administeredtopically as a formulation ranging from about 0.01% (w/v) to about 3%(w/v), e.g., 0.02%, 0.03%, 0.04%, 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 1%, 1.2%, 1.3%, 1.5%, 1.7%, 2%, 2.2%, 2.5%,2.8%, or another topical concentration from about 0.01% (w/v) to about3% (w/v). In some embodiments, the inhibitor concentration is about0.02% (w/v) to about 2%. In preferred embodiments the topicalconcentration is about 0.1% to about 0.5%.

In some embodiments, the subject to be treated receives multipleadministrations of at least one inhibitor within a dosing period.Accordingly, in some embodiments, where administration of an agent thatinhibits the Hh signalling pathway is by systemic, intraperitoneal, orintrapleural administration, the total aggregate dose per dosing periodfor an inhibitor ranges from about 0.5 mg/kg to about 25 mg/kg per totalaggregate dose per dosing period, e.g., 0.6 mg/kg, 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 18 mg/kgor another dose ranging from about 0.5 mg/kg to about 25 mg/kg per totalaggregate dose. In some embodiments, the total aggregate dose per dosingperiod is about 1 mg/kg to about 10 mg/kg per total aggregate dose perdosing period.

In some preferred embodiments the at least one agent to inhibit the Hhsignalling pathway is the small molecule inhibitor Vismodegib. In someembodiments, where administration of Vismodegib is systemic, the doseconcentration in circulation is about 10 μM to about 80 μM, e.g., 15 μM,20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, oranother concentration in circulation from about 10 μM to about 80 μM. Insome preferred embodiments, the dose concentration in circulation is atleast about 20 μM to about 40 μM, e.g., 25 μM, 30 μM, 35 μM, or anotherconcentration in circulation from at least about 20 μM to about 40 μM.

In some embodiments, where administration of Vismodegib isintralesional, the dose concentration is about 10 μM to about 40 μM perlesion, e.g., 15 μM, 20 μM, 25 μM, 30 μM, 35 μM per lesion, or anotherdose concentration from about 10 μM to about 40 μM. In other embodimentsVismodegib is administered intralesionally at a dose ranging from about0.0025 mg/lesion to about 0.3 mg/lesion, e.g., 0.005 mg/lesion, 0.0075mg/lesion, 0.01 mg/lesion, 0.02 mg/lesion, 0.025 mg/lesion, 0.04mg/lesion, 0.05 mg/lesion, 0.07 mg/lesion, 0.1 mg/lesion, 0.15mg/lesion, 0.2 mg/lesion, 0.25 mg/lesion, or another intralesional dosefrom about 0.0025 mg/lesion to about 0.3 mg/lesion. In preferredembodiments intralesional administration of Vismodegib is in a doseranging from about 0.0025 mg/lesion to about 0.05 mg/lesion.

In some embodiments Vismodegib is administered by a systemic (e.g.,oral), intraperitoneal, or intrapleural route at a dose of about 0.1mg/kg to about 12 mg/kg per administration, e.g., 0.20 mg/kg, 0.5 mg/kg,1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 10 mg/kg, or another doseranging from about 0.1 mg/kg to about 12 mg/kg. In some preferredembodiments, the dose of Vismodegib is about 2 mg/kg to about 5 mg/kg.In some preferred embodiments Vismodegib administered orally with a doseof about 150 mg to about 500 mg daily, e.g., 160 mg, 170 mg, 200 mg, 250mg, 300 mg, 350 mg, 400 mg, or another oral dose from about 150 mg toabout 500 mg daily.

In some embodiments, where a subject is to be treated for a diseaseamenable to intralesional administration (e.g., basal cell carcinoma orcutaneous warts), both an inhibitor of the Hh signalling pathway and atype II or type I interferon are administered by an intralesional route.In exemplary preferred embodiments a non-replicative recombinantadenovirus is used for expression of interferon gamma, and isadministered in a dose ranging from about 5×10¹⁰ viral particles/lesionto about 3×10¹¹ viral particles/lesion about 2 to 5 times per week, anda small molecule inhibitor of the Hh signalling pathway is administeredorally in a dose ranging from about 2 mg/kg to about 5 mg/kg daily. Insome embodiments the just-mentioned treatment is repeated for up to fourweeks.

Pharmaceutical Compositions

Any of the therapeutic agents described herein can be formulated eitheralone or in combined pharmaceutical compositions as described herein foradministration to a subject via any conventional means including, butnot limited to, intralesional, parenteral (e.g., intravenous,subcutaneous, intramuscular, intraperitoneal, or intrapleural), oral, ortransdermal administration routes.

Thus, in some embodiments provided herein is pharmaceutical compositionfor use in treatment of a disease characterised by aberrant cellproliferation, the pharmaceutical composition comprising: (i) at leastone type II interferon and/or type I interferon, or a polynucleotideencoding a type II interferon and/or type I interferon; and (ii) atleast one inhibitor of the Hedgehog (Hh) signalling pathway. In someembodiments the pharmaceutical composition also include an inhibitor ofWNT signalling.

Also provided herein is a pharmaceutical composition for use intreatment of a disease characterised by aberrant cell proliferation, thepharmaceutical composition comprising: (i) at least one agent thatincreases activation of a receptor of at least one type II interferonand/or type I interferon; and (ii) at least one agent which inhibits theHedgehog (Hh) signalling pathway. In some embodiments of thepharmaceutical (i) includes at least one type II interferon and/or typeI interferon, or a polynucleotide encoding a type II interferon and/ortype I interferon, or an agonist for a receptor of at least one type IIinterferon and/or type I interferon. In some embodiments thepharmaceutical composition includes a recombinant virus comprising thepolynucleotide encoding the type II interferon and/or type I interferonfor expression in a subject. In some preferred embodiments theinterferon (or the interferon to be expressed) is interferon gamma.

Therapeutic agents can be formulated into any suitable dosage form,including but not limited to, injectable formulations, aqueous oraldispersions, liquids, mists, gels, syrups, elixirs, slurries,suspensions and the like, for oral ingestion by a patient to be treated,solid oral dosage forms, controlled release formulations, lyophilizedformulations, tablets, powders, pills, dragees, capsules, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the therapeutic agentsdescribed herein, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipientsinclude, for example, fillers such as sugars, including lactose,sucrose, mannitol, or sorbitol; cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents may be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Pharmaceutical solid dosage forms can include, in addition to thetherapeutic agents, one or more pharmaceutically acceptable additivessuch as a compatible carrier, binder, filling agent, suspending agent,flavoring agent, sweetening agent, disintegrating agent, dispersingagent, surfactant, lubricant, colorant, diluent, solubilizer, moisteningagent, plasticizer, stabilizer, penetration enhancer, wetting agent,anti-foaming agent, antioxidant, preservative, or one or morecombination thereof.

Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Suitable filling agents for use in the solid dosage forms describedherein include, but are not limited to, lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, hydroxypropylmethycellulose(HPMC), hydroxypropylmethycellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose,xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethyleneglycol, and the like.

In order to release the therapeutic agents from a solid dosage formmatrix as efficiently as possible, disintegrants are often used in theformulation, especially when the dosage forms are compressed withbinder. Disintegrants help rupturing the dosage form matrix by swellingor capillary action when moisture is absorbed into the dosage form.Suitable disintegrants for use in the solid dosage forms describedherein include, but are not limited to, natural starch such as cornstarch or potato starch, a pregelatinized starch such as National 1551or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, acellulose such as a wood product, methylcrystalline cellulose, e.g.,Avicel® PH101, Avicel®PH102, Avicel®PH105, Elcema® P100, Emcocel®,Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose,or a cross-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrospovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose, methylcellulose(e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USPPharmacoat-603, hydroxypropylmethylcellulose acetate stearate (AqoateHS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g.,Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystallinecellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesiumaluminum silicate, polysaccharide acids, bentonites, gelatin,polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone,starch, pregelatinized starch, tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such asacacia, tragacanth, ghatti gum, mucilage of isapol husks, starch,polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL,Polyplasdone®XL-10, and Povidone®K-12), larch arabogalactan, Veegum®,polyethylene glycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder. Formulators skilled in art can determine the binder level forthe formulations, but binder usage level of up to 70% in tabletformulations is common.

Suitable lubricants or glidants for use in the solid dosage formsdescribed herein include, but are not limited to, stearic acid, calciumhydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal andalkaline earth metal salts, such as aluminum, calcium, magnesium, zinc,stearic acid, sodium stearates, magnesium stearate, zinc stearate,waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol or a methoxypolyethylene glycolsuch as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol,sodium oleate, glyceryl behenate, glyceryl palmitostearate, glycerylbenzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described hereininclude, but are not limited to, sugars (including lactose, sucrose, anddextrose), polysaccharides (including dextrates and maltodextrin),polyols (including mannitol, xylitol, and sorbitol), cyclodextrins andthe like.

Suitable wetting agents for use in the solid dosage forms describedherein include, for example, oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodiumoleate, sodium lauryl sulfate, magnesium stearate, sodium docusate,triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described hereininclude, for example, sodium lauryl sulfate, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms describedhere include, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in solid dosageforms described herein. The amounts of such additives can be readilydetermined by one skilled in the art, according to the particularproperties desired.

Liquid formulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

In addition to the additives listed above, the liquid formulations canalso include inert diluents commonly used in the art, such as water orother solvents, solubilizing agents, and emulsifiers. Exemplaryemulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodiumdoccusate, cholesterol, cholesterol esters, taurocholic acid,phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

Formulations suitable for intramuscular, subcutaneous, or intravenousinjection may include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, therapeutic agents described herein may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological saline buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art. For other parenteralinjections, appropriate formulations may include aqueous or nonaqueoussolutions, preferably with physiologically compatible buffers orexcipients. Such excipients are generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the therapeutic agents in water-solubleform. Additionally, suspensions of the therapeutic agents may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of atherapeutic agent to allow for the preparation of highly concentratedsolutions. Alternatively, the therapeutic agent may be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use. The therapeutic agents described herein may be inunit dosage forms suitable for single administration of precise dosages.In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of one or more therapeutic agents. Theunit dosage may be in the form of a package containing discretequantities of the formulation. Non-limiting examples are packagedtablets or capsules, and powders in vials or ampoules. Aqueoussuspension compositions can be packaged in single-dose non-reclosablecontainers. Alternatively, multiple-dose reclosable containers can beused, in which case it is typical to include a preservative in thecomposition. By way of example only, formulations for parenteralinjection may be presented in unit dosage form, which include, but arenot limited to ampoules, or in multi-dose containers, with an addedpreservative.

Also provided herein are controlled release pharmaceutical compositions.Controlled release refers to the release of therapeutic agents from adosage form in which they are incorporated according to a desiredprofile over an extended period of time. Controlled release profilesinclude, for example, sustained release, prolonged release, pulsatilerelease, and delayed release profiles. In contrast to immediate releasecompositions, controlled release compositions allow delivery of an agentto a subject over an extended period of time according to apredetermined profile. Such release rates can provide therapeuticallyeffective levels of a therapeutic agent for an extended period of timeand thereby provide a longer period of pharmacologic response whileminimizing side effects as compared to conventional rapid release dosageforms. Such longer periods of response provide for many inherentbenefits that are not achieved with the corresponding short acting,immediate release preparations.

The controlled release pharmaceutical compositions provided herein allowthe release profile of an active agent within the combinationformulation to be customised so that release of one or more of theseactive agents occurs over a preferred time interval. In some preferredembodiments, the agent that inhibits the Hh signalling pathway in thecontrolled release pharmaceutical composition is a small moleculeinhibitor of Hh signalling pathway.

In some embodiments the pharmaceutical composition comprises from about150 mg to about 3000 mg of a small molecule inhibitor of the Hhsignalling pathway per day, e.g., 200 mg, 300 mg, 600 mg, 800 mg, 1000mg, 1200 mg, 1600 mg, 1800 mg, 2000 mg, 2400 mg, 2800 mg, or anotherdose from about 100 mg to about 3000 mg of the small molecule inhibitor.In some embodiments the controlled release pharmaceutical compositioncomprises from about 200 mg to about 1400 mg of the small moleculeinhibitor. In other embodiments the controlled release pharmaceuticalcomposition comprises from about 600 mg to about 1000 mg of the smallmolecule inhibitor.

In some embodiments, one or more of the active agents is released over atime period ranging from about one hour to about five weeks, e.g., 2hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours,2 days, 3 days, 5 days, 1 week, 10 days, 2 weeks, 18 days, 3 weeks, 4weeks, or another period from about one hour to about five weeks.

In some embodiments, the controlled release profile has a release ratehigher at the beginning of the release period following administration,and then decreases over time (first order release kinetics). In otherembodiments, the release rate progressively increases over the releaseperiod following administration. In preferred embodiments, the releaseprofile remains relatively constant over the entire release periodfollowing administration until all of the active agent is released (zeroorder release kinetics).

In preferred embodiments the release profile of a small moleculeinhibitor of the Hh signalling pathway upon administration of thecontrolled release pharmaceutical composition is adapted to avoidinduction of at least one adverse event in the human subject. In someembodiments, the release rate of a small molecule inhibitor of the Hhsignalling pathway is about 0.5% of the total dose/day to about 10% ofthe total dose per day, e.g., 0.6%, 0.8%, 1%, 1.1%, 1.2%, 1.5%, 1.8%,2%, 2.5%, 2.75%, 3%, 3.5%, 4%, 6%, 7%, 8%, 9% or another percentage ofthe total dose per day from about 0.5% to about 10% per day.

In some embodiments, the rate of release (as a percentage of total dose)of a small molecule inhibitor in the controlled release formulation isdistinct from that the release profile of another active agent in theformulation (e.g., a recombinant virus, a polynucleotide, or a purifiedprotein or peptide).

In some embodiments, where the controlled release pharmaceuticalcomposition includes interferon gamma, the interferon gamma is releasedat a rate of about 1 μg/day to about 10 μg/day for systemic delivery,e.g., 2 μg/day, 3 μg/day, 4 μg/day, 5 μg/day, 7 μg/day, 8 μg/day, oranother release rate for systemic administration of purified interferongamma ranging from about 1 μg/day to about 10 μg/day.

In some embodiments, where the controlled release pharmaceuticalcomposition includes interferon gamma, the interferon gamma is releasedat a rate of about 5 ng/day to about 200 ng/day for localized delivery,e.g., implantation within a tumour, e.g., 10 ng/day, 20 ng/day, 30ng/day, 50 ng/day, 75 ng/day, 100 ng/day, 125 ng/day, 150 ng/day, 175ng/day, or another release rate for local administration ranging fromabout 10 ng/day to about 200 ng/day.

Where at least one recombinant virus is to be included in apharmaceutical composition described herein, such pharmaceuticalcompositions described herein may comprise a range of viral titers,expressed as a 50% tissue culture infective dose (TCID₅₀)/m1 and/orviral particles (vp)/ml, depending on a number of considerationsincluding the condition to be treated, the subject to be treated, adesired release rate and the desired treatment period per dose. In someembodiments the pharmaceutical compositions described herein have atiter of about 1×10⁹ TCID₅₀/ml to about 3×10¹⁰ TCID₅₀/ml, e.g., 1.5×10⁹TCID₅₀/ml, 1.8×10⁹ TCID₅₀/ml, 2.0×10⁹ TCID₅₀/ml, 3.0×10⁹ TCID₅₀/ml,4.0×10⁹ TCID₅₀/ml, 5.0×10⁹ TCID₅₀/ml, 5.5×10⁹ TCID₅₀/ml, 6.0×10⁹TCID₅₀/ml, 6.5×10⁹ TCID₅₀/ml, 7.0×10⁹ TCID₅₀/ml, 7.5×10⁹ TCID₅₀/ml,8.0×10⁹ TCID₅₀/ml, 8.5×10⁹ TCID₅₀/ml, 9.0×10⁹ TCID₅₀/ml, 1.0×10¹⁰TCID₅₀/ml, 1.5×10¹⁰ TCID₅₀/ml, 2.0×10¹⁰ TCID₅₀/ml, 2.5×10¹⁰ TCID₅₀/ml,or another TCID₅₀/ml value from about 1×10⁹ TCID₅₀/ml to about 3×10¹⁰TCID₅₀/ml. In some preferred embodiments, the TCID₅₀/ml is about 4×10⁹TCID₅₀/ml to 8×10⁹ TCID₅₀/ml.

In some embodiments the equivalence of vp/TCID₅₀ is approximately 20 to100 vp/TCID₅₀. Accordingly, in some embodiments the pharmaceuticalcompositions described herein have a titer of about 2×10¹⁰ vp/ml toabout 3×10¹² vp/ml, e.g., 2×10¹⁰ vp/ml, 3×10¹⁰ vp/ml, 4×10¹⁰ vp/ml,5×10¹⁰ vp/ml, 6×10¹⁰ vp/ml, 7×10¹⁰ vp/ml, 8×10¹⁰ vp/ml, 9×10¹⁰ vp/ml1×10¹¹ vp/ml, 2×10¹¹ vp/ml, 3×10¹¹ vp/ml, 4×10¹¹ vp/ml, 5×10¹¹ vp/ml,6×10¹¹ vp/ml, 7×10¹¹ 8×10¹¹ vp/ml, 9×10¹¹ vp/ml, 1×10¹² vp/ml, 2×10¹²vp/ml, or another titer from about 2×10¹⁰ vp/ml to about 3×10¹² vp/ml.In some preferred embodiments the titer is from about 3×10¹⁰ vp/ml toabout 8×10¹¹ vp/ml. In other preferred embodiments the titer of thepharmaceutical composition is about 3×10¹⁰ viral particles/ml to about5×10¹² viral particles/ml.

Suitable controlled release matrices for controlled releasepharmaceutical compositions have been described in art. In someembodiments, the SiO₂ matrix hydrogel is a bioresorbable sol-gel derivedTetraethyl orthosilicate (AKA “tetrathoxysilane” or “TEOS”) Si (OC₂H₅)₄matrix gel (“SiO₂ matrix gel”). This technology has been commercialisedby DelSiTech Ltd (Turku, Finland). Such a bioresorbable SiO₂ matrix gelis useful for sustained delivery of active therapeutic agents includingsmall molecule drugs and recombinant viruses as described ininternational patent application publications WO2005082781 entitled“Method for Preparing Adjustably Bioresorbable Sol-Gel Derived SiO₂” andWO2007135224 entitled “Method for Storing Silica-Based Material, PackageProduced with the Method, and Use of Package for Packaging ofSilica-Based Products.” This technology has been commercialised byDelSiTech Ltd (Turku, Finland).

In brief, the SiO₂ matrix gel sol-gel is prepared by the sol-gel processwherein the SiO₂ matrix gel is prepared from a sol comprising SiO₂ thathas turned to a gel. Sol-gel derived SiO₂ is typically prepared fromalkoxides or inorganic silicates that via hydrolysis form a sol thatcontains either partly hydrolysed silica species or fully hydrolysedsilicic acid. Consequent condensation reactions of SiOH containingspecies lead to formation of larger silica species with increasingamount of siloxane bonds. Furthermore, the species aggregate, formnanosized particles and/or larger aggregates until a gel is formed. Inthe form of a gel, the solid state dominates, but the system stillcontains varying amounts of liquids and the material is typically softand viscoelastic before drying and hard and brittle if it is extensivelydried. In the form of a sol, liquid state dominates, but the systemcontains varying amounts of solid phase(s) and the material is stillflowable. The time from when the SiO₂ sol is prepared until the solturns to a gel is referred to as sol ageing time. Spontaneous dryingtypically occurs when the sol is aged so that the system allowsevaporation in ambient conditions. Generation of the controlled releasepharmaceutical composition is achieved by adding to the sol, before gelformation, the desired amounts of the active therapeutic agents to beincluded in the pharmaceutical composition (e.g., recombinant virusexpressing interferon gamma and a small molecule inhibitor of the Hhsignalling pathway). As an end result of this process, a pharmaceuticalcomposition is obtained which contains a SiO₂ matrix hydrogel thatcontains one or more recombinant viruses for expression of a Type I orType II interferon; an agent for inhibiting the Hh signalling pathway;SiO₂ matrix hydrogel; and silica microparticles, where the recombinantviruses and the agent for inhibiting the Hh signalling pathway areinterspersed within the SiO₂ matrix hydrogel.

Release rates of the active agents in SiO₂ gel-based controlled releasepharmaceutical compositions can be adjusted as needed. Generally themaximum dissolution rate of the SiO₂ gel matrix and release rate of theactive agents occurs for SiO₂ gels having a molar ratio of water toalkoxide of about 2, with ratios lower or higher than this resulting inslower dissolution and release rates. Further, It should also be notedthat large amounts of active agent comprised within the SiO₂ gel matrixincreases dissolution of the matrix and the release rate(s) of theactive agents.

The controlled release pharmaceutical compositions can be prepared asnano- and microspheres mainly for oral, parenteral, pulmonary, topical,transdermal and surgically implantable administration.

In some embodiments the rate of recombinant virus release (rate ofdissolution) observed for a pharmaceutical composition described hereinoccurs at approximately ten times the rate in vitro than it does invivo.

In exemplary, non-limiting embodiments, the pH of a water and tetraethylorthosilicate (TEOS) mixture at an initial molar ratio of about 100:1 to150:1 is adjusted to pH 2 with hydrochloric acid and vigorously stirredat room temperature for 25 min. The pH of the sol is then adjusted tothe desired pH (6, 6.5 or 7) by adding 0.1 M NaOH. The sol is cooled inan ice-water bath and the desired amounts of the active agents to beincluded are added.

In some embodiments the SiO₂ matrix hydrogel in the pharmaceuticalcomposition comprise water and TEOS in a final molar ratio of about 5:1to about 4,000:1, e.g., 10:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1,300:1, 400:1, 500:1, 750:1, 1,000:1, 2,000:1, 3,000:1, or another finalmolar ratio of water to TEOS from about 50:1 to about 700:1, or about5:1 to about 1,000:1. In some preferred embodiments the final molarratio of water to TEOS is about 400:1.

Many other types of controlled release systems known to those ofordinary skill in the art and are suitable for use with the formulationsdescribed herein. Examples of such delivery systems include, e.g.,polymer-based systems, such as polylactic and polyglycolic acid,plyanhydrides and polycaprolactone; porous matrices, nonpolymer-basedsystems that are lipids, including sterols, such as cholesterol,cholesterol esters and fatty acids, or neutral fats, such as mono-, di-and triglycerides; hydrogel release systems; silastic systems;peptide-based systems; wax coatings, bioerodible dosage forms,compressed tablets using conventional binders and the like. See, e.g.,U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509, 5,461,140, 5,456,923,5,516,527, 5,622,721, 5,686,105, 5,700,410, 5,977,175, 6,465,014 and6,932,983.

In some embodiments a pharmaceutical composition described herein isgenerated as a depot formulation.

EXAMPLES Example 1—Anti-Tumour Efficacy of a Combination Therapy ofAdenovirus Expressing IFNγ and Vismodegib

Vismodegib is a hedgehog inhibitor approved for use in BCC. Theanti-tumour efficacy of the combination of Ad5-mIFNgamma with Vismodegibwill be evaluated in a number of murine tumour models. Suitable modelsinclude the murine melanoma cell line, B16-F0 and later in subcutaneoustumour graft models 4T1 (breast tumour line), CT26 (mouse coloncarcinoma line) and human tumour xenograft MDA-MB-231 (human breastcancer line).

Experimental Design

2×10⁵ tumour cells will be implanted subcutaneously in C57BL/6 mice togenerate tumours. Once tumours are 3-6 mm in diameter, 1×10¹⁰ viralparticles (VPs)/tumour of Ad-5-mIFNγ, Vismodegib (50 mg or 100 mg/kg byoral gavage), or both will be administered to various experimentalgroups according to the schedule shown in Table 1. In Table 1, day “0”denotes the initial day of treatment. Vismodegib will be administered inDMSO or PEG 400/5% dextrose in water (75:25 v/v).

Mice to be monitored by caliper measurement for tumour growth everyother day and survival. End points will be determined according toapproved animal ethics requirements including maximum tumour sizeallowed and animal sacrifice if any signs of distress are apparent.

Data Analysis

Data will be recorded and represented in individual tumour growth curvesof all animals in groups as mean±error as well as statisticalsignificance. Survival will be plotted as Kaplan-Meier survival curvesand statistical comparison will be performed using a log-rank test.

Tumours from 2 mice/group (point of removal to be determined) from abovefor mRNA isolation, formalin fixation and/or multiparameter flowcytometric analysis of tumour infiltrating cells.

TABLE 1 Treatment Schedule for Administration of Ad-5-mIFN-gamma andVismodegib Combination Treatment in a Murine Tumour Model Regimen Agent(Ad-mIFNγ) Agent (Vismodegib) Schedule Schedule Group N Dose (Day) RouteDose (Day) Route Comment 1 10 Vehicle 0, 1, 2  IT Vehicle 0-6 OralTumour response and survival 2 10 Vehicle 0, 1, 2  IT  50 mg/kg 0-6 Oralwill be evaluated before end 3 10 10¹⁰ VP 0, 1, 2  IT Vehicle 0-6 Oralof 1^(st) treatment cycle and 4 10 10¹⁰ VP 0, 1, 2  IT  50 mg/kg 0-6Oral will assess if another course of treatment will be initiated (D7,8, 9 Ad-mIFNγ and Vismo D7-D13) 5 10 Vehicle 0, 7, 14 IT Vehicle3x/weekx3 Oral 6 10 Vehicle 0, 7, 14 IT 100 mg/kg 3x/weekx3 Oral 7 1010¹⁰ VP 0, 7, 14 IT Vehicle 3x/weekx3 Oral 8 10 10¹⁰ VP 0, 7, 14 IT 100mg/kg 3x/weekx3 Oral

Example 2—Combination Therapy of Adenovirus Expressing IFNγ andVismodegib Reduces Tumour Growth in Mice

Methods

Mice

Female B6D2F1/J mice (Jackson Laboratories) were ten weeks old and hadan individual body weight (BW) range of 19.4 to 29.9 g on Day 1 of thestudy.

Tumour Cell Culture

The B16F10 murine colon carcinoma cell line was maintained in DMEMmedium containing 10% fetal bovine serum, 2 mM glutamine, 100 units/mLsodium penicillin G, 25 μg/mL gentamicin, and 100 μg/mL streptomycinsulfate. The tumour cells were cultured in tissue culture flasks in ahumidified incubator at 37° C., in an atmosphere of 5% CO₂ and 95% air.

In Vivo Tumour Implantation and Measurement

B16F10 cells were harvested during log phase growth and resuspended inPBS. Each mouse was injected subcutaneously in the right flank with5×10⁵ cells (in a 0.1 mL cell suspension). Tumours were calipered in twodimensions to monitor growth as their mean volume approached the desired30 to 60 mm³ range. Tumour size, in mm³, was calculated from:

${{Tumour}\mspace{14mu}{Volume}} = \frac{w^{2} \times l}{2}$where w=width and l=length, in mm, of the tumour. Tumour weight can beestimated based on the assumption that 1 mg is equivalent to 1 mm³ oftumour volume.

Eight days after tumour cell implantation, on Day 1 of the study,animals were sorted into eight groups (n=10 per group) according totumour volume, with individual tumour volumes ranging from 32 to 63 mm³and group mean tumour volumes ranging from 41 to 42 mm³. Tumourprogression was monitored using caliper measurements three times weeklyfor the duration of the study.

Therapeutic Agents

Vismodegib and Ad5-mIFNg (stock concentration 3.3×10¹² viralparticles/mL) were stored at −80° C. prior to formulation. On each dayof dosing, Vismodegib was dissolved in vehicle 2 (75% PEG400:25% D5W (5%dextrose in water)) to obtain 5 mg/mL and 10 mg/mL dosing solutionswhich delivered a dose of 50 mg/kg and 100 mg/kg when administered in avolume of 10 mL/kg (0.2 mL per 20 g mouse), adjusted to the body weightof each animal.

On the first day of dosing, Ad5-mIFNg was thawed rapidly and diluted invehicle 1 (1 M saccharose, 54 mg/L Tween® 80, 10 mM Tris, 1 mM MgCl₂,150 mM NaCl, pH 8) to obtain dosing solutions of 5×10¹¹ particles/mL and2×10¹¹ particles/mL, which delivered 1×10¹⁰ particles/animal when dosedin a fixed volume of 20 μL and 50 μL per animal, respectively.

Treatment

On Day 1 of the study, female mice bearing established B16F10 tumourswere sorted into eight groups (n=10 per group) and began dosingaccording to the treatment plan summarized in Table 2. Vehicle 1 andAd5-mIFNg (1×10¹⁰ particles per animal) were administeredintratumourally (itu) in a fixed volume of either 20 μL or 50 μL;vehicle 2 and Vismodegib were administered orally (po) in a volume of 10mL/kg (0.2 mL per 20 g mouse), adjusted to the body weight of eachanimal. The groups were as follow:

Group 1 served as the control group and received 20 μL vehicle 1 ituonce a day (qd) for three days (qd×3) and vehicle 2 po qd×7.

Group 2 received 20 μL vehicle 1 itu qd×3 and 50 mg/kg vismodegib poqd×7.

Group 3 received 1×10¹⁰ particles/animal Ad5-mIFNg in 20 μL itu qd×3 andvehicle 2.

Group 4 received 1×10¹⁰ particles/animal Ad5-mIFNg 1 itu qd×3 and 50mg/kg vismodegib 2 po qd×7.

Group 5 received 20 μL vehicle 1 itu once a week for a week (qwk×1)followed by 50 μL vehicle 1 itu qwk×1 (starting on Day 8) in addition tovehicle 2 po three times a week (tiwk) to Day 10.

Group 6 received 20 μL vehicle 1 itu once a week for a week (qwk×1)followed by 50 μL vehicle 1 itu qwk×1 (starting on Day 8) and 100 mg/kgVismodegib in vehicle 2 po tiwk to Day 13.

Group 7 received 20 μL containing 1×10¹⁰ particles/animal Ad5-mIFNg ituonce a week for a week (qwk×1) followed by 50 μL containing 1×10¹⁰particles/animal Ad5mIFNg itu qwk×2 (starting on Day 8) and vehicle 2three times a week for three weeks (tiwk×3).

Group 8 received 20 μL containing 1×10¹⁰ particles/animal Ad5-mIFNg ituonce a week for a week (qwk×1) followed by 50 μL containing 1×10¹⁰particles/animal Ad5mIFNg itu qwk×2 (starting on Day 8) and 100 mg/kgvismodegib po three times a week for three weeks (tiwk×3).Endpoint and Tumour Growth Delay (TGD) Analysis

Tumours were measured using calipers three times per week, and eachanimal was euthanized when its tumour reached the pre-determined tumourvolume endpoint of 1500 mm³ or on the last day of the study (Day 45).Animals that exited the study for tumour volume endpoint were documentedas euthanized for tumour progression (TP), with the date of euthanasia.

Results

B16F10 tumour-bearing mice were treated as indicated in Table 2 andtumour growth monitored by caliper measurement. As shown in FIG. 1,treatment with oral Vismodegib qdx7 (G2) or tiwkx3 (G6) did notsignificantly reduce tumour growth in mice compared to the controlvehicle treated groups. In fact, none of the mice in these groups werealive after day 12 (culled at ethical end point). For comparison oftumour inhibition, mean tumour volumes were compared when there were atleast four mice alive out of an initial eight mice/group.

Ad5-mIFNg administered intratumourally either qdx3 (G3) or qwkx1 (G7)significantly inhibited tumour growth compared to controls (G1 and G6).There was no significant difference in therapeutic efficacy in thecombination of treatment dose regimen Ad5-mIFNg (qwkx1) alone andvehicle 2 (G7) versus Ad5-mIFNg (qwkx1) in combination with Vismodegib(tiwkx3) (G8). In contrast, a combination treatment dosing of Ad5-mIFNg(qdx3) and Vismodegib (qd×7) (G4) retarded the tumour growth more thanthe individual treatments (G3 and G2). On day 36, 50% of the mice (4 outof 8 mice) in G4 were still alive compared to 1 out of 8 mice treatedwith Ad5-mIFNg only qd×3 (G3) or no mice from the group treated withVismodegib only (G2) qd×7. On day 20 the mean tumour size of the (G4)combination treatment group was 30% that of the tumours of mice treatedwith Ad5-mIFNg alone (G3).

TABLE 2 Protocol Design for the B16F10-e357 Study Treatment Regimen 1Treatment Regimen 2 Treatment Regimen 3 Group Agent u/animal RouteSchedule Agent u/animal Route Schedule Agent u/animal Route Schedule 1Vehicle 1 — itu  qd × 3 Vehicle 2 — po qd × 7 — — — — 2 Vehicle 1 — itu qd × 3 Vismodegib 50^(a) po qd × 7 — — — — 3 Ad5- 1 × 10¹⁰ itu  qd × 3Vehicle 2 — po qd × 7 — — — — mIFNg 4 Ad5- 1 × 10¹⁰ itu  qd × 3Vismodegib 50^(a) po qd × 7 — — — — mIFNg 5 Vehicle 1 — itu qwk × 1Vehicle 1 — itu qwk × 2 (start Vehicle 2 — po tiwk × 3 on Day 8) 6Vehicle 1 — itu qwk × 1 Vehicle 1 — itu qwk × 2 (start Vismodegib100^(a) po tiwk × 3 on Day 8) 7 Ad5- 1 × 10¹⁰ itu qwk × 1 Ad5- 1 × 10¹⁰itu qwk × 2 (start Vehicle 2 — po tiwk × 3 mIFNg mIFNg on Day 8) 8 Ad5-1 × 10¹⁰ itu qwk × 1 Ad5- 1 × 10¹⁰ itu qwk × 2 (start Vismodegib 100^(a)po tiwk × 3 mIFNg mIFNg on Day 8) Table 2 displays the study design asof Day 1 of the study Vehicle 1 = Vehicle for Ad5-mIFNg-ASCND Saccharose1M, Tween80 54 mg/l, Tris 10 mM, MgCl₂ 1 mM, NaCl 150 mM, pH 8 Vehicle 2= Vehicle for Vismodegib- ASCND PEG 400/5% dextrose in water (75:25 v/v)^(a)mg/kg

Example 3—Combination Therapy of Adenovirus Expressing IFNγ andVismodegib in Basal Cell Nevus Syndrome Patients

In an ongoing clinical trial (26 week duration), patients presentingwith multiple low risk basal cell carcinomas in sporadic or basal cellnevus syndrome patients received Vismodegib (150 mg, oral) daily forfour weeks and three single intralesional injections of ASN-002 of 1×10″VP/lesion (one injection per week at weeks 3-5). Patients were monitoredfor treatment response through week 26 of the study (i.e.,post-treatment period of 19 weeks).

Over this period a total of 23 lesions was monitored (including singleor multiply injected lesions, and non-injected lesions). At week 25, aloco-regional complete histological clearance (CHC) of >50% was observedin response to this combination treatment. In comparison a previousstudy using ASN-002 treatment alone, loco-regional CHC of 0% wasobserved at week 18. In a study by Sofen et al. (2015), operable basalcell carcinoma patients receiving a daily dose of 150 mg of Vismodegibover a period of 12 weeks exhibited a loco-regional CHC of 16%.

Thus, as expected based on the methods disclosed herein, the results ofthis ongoing study to date underscore that combination treatment withVismodegib and ASN-002 provides a superior therapeutic response comparedto monotherapy with either of these therapeutic agents.

The present application claims priority from AU2020900585 filed 28 Feb.2020, and AU 2020900813 filed 17 Mar. 2020, the entire contents of bothof which are incorporated herein by reference.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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The invention claimed is:
 1. A method of treating a human subjectsuffering from basal cell carcinoma (BCC), the method comprisingadministering to the human subject a therapeutically effective amountof: (i) a recombinant DNA virus for expression of interferon gamma byintralesional or perilesional injection into one or more BCC-relatedlesions; and (ii) Vismodegib or Sonidegib, wherein the subject istreated over a period of one week to twelve weeks.
 2. The method ofclaim 1, wherein the recombinant DNA virus is an adenovirus.
 3. Themethod of claim 1, comprising administering at least 5×10¹⁰ viralparticles (vp) per lesion.
 4. The method of claim 3, comprisingadministering from 5×10¹⁰ vp per lesion to 1.5×10¹¹ vp per lesion. 5.The method of claim 1, comprising administration of Sonidegib.
 6. Themethod of claim 1, comprising administration of Vismodegib.
 7. Themethod of claim 1, wherein the Vismodegib or Sonidegib is administeredat a dose of 50 mg/day to 500 mg/day.
 8. The method of claim 7, whereinthe dose is 150 mg/day.
 9. The method of claim 1, wherein the Vismodegibor Sonidegib is administered systemically.
 10. The method of claim 9,wherein the Vismodegib or Sonidegib is administered orally.